We have been developing a system for real-time single molecule DNA sequencing. System components include novel "charge-switched" nucleotides, an adapted DNA polymerase, a method for isolating and handling single DNA molecules, a microfluidics flowcell for sorting molecules by charge, a TIR (total internal reflection) optical system for single-molecule detection in four spectral channels, and software algorithms for single molecule detection and system control. This proposal will develop data analysis techniques and algorithms to perform single molecule base calling, in an automated fashion, from CCD images. We will also identify a set of measurable image parameters that may influence base call accuracy. In Phase II, we will use statistical methods to determine which parameters are most strongly correlated to a correct base call, so that quality values may be developed. These values will be the single-molecule sequencing analogs of the quality scores used in PHRED and PHRAP from conventional DNA sequencing. A key advantage of single-molecule sequencing is the elimination of cloning and the laboratory infrastructure associated with high-throughput operations. This, along with reduced reagent consumption, results in sequencing costs orders of magnitude lower compared to existing methods. Read lengths will be tens of kilobases to simplify shotgun sequence assembly and preserve haplotype information. Applications include whole-genome sequencing, SNPs, haplotyping, genotype-trait associations, long-read SAGE for expression profiling, analysis of alternative mRNA splicing patterns, and comparative genomics within or between species. Fields-of-use include research, diagnostics and personalized medicine.